Determination of Allicin in Allium Sativum Using High Performance Liquid Chromatography and Study of Genotoxic Effect on Human Leukocytes
Total Page:16
File Type:pdf, Size:1020Kb
Vol 8, Issue 6, 2015 ISSN - 0974-2441 Research Article DETERMINATION OF ALLICIN IN ALLIUM SATIVUM USING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY AND STUDY OF GENOTOXIC EFFECT ON HUMAN LEUKOCYTES NISHU SEKAR, RAJIV SUNDARAMOORTHY, SRISTI MAJUMDAR, KRISHNAKSHI BHUYAN, JYOTIREKHA DAS, ABILASH VALSALA GOPALAKRISHNAN* Division of Biomolecules and Genetics, School of Biosciences and Technology, VIT University, Vellore, Tamil Nadu, India. Email: [email protected] Received: 21 July 2015, Revised and Accepted: 07 September 2015 ABSTRACT Objectives: Allicin is an organosulfur compound featuring thiosulfinate functional group. The compound is formed in garlic after tissue damage, by the action of enzyme alliinase on alliin. This study involves extraction of allicin from garlic using high-performance liquid chromatography (HPLC) and study of it is the genotoxic effect on human leukocytes. Methods: A simple and rapid reverse phase HPLC was used for the extraction of allicin. Quality allicin, in conjugation with chymosin, was used to study its genotoxic effect on leukocytes. Results: Garlic oil and garlic extract showed similar retention time, and we measured the products using genotoxic effects in human leukocyte culture and it shows statistically not significant. Conclusion: This study suggested that to take a lower concentration of garlic extracts benefits for health and these findings useful for further research. Keywords: Allium sativum, Leukocytes, Chromosome, Genotoxic, High-performance liquid chromatography. INTRODUCTION METHODS Garlic (Allium sativum) possesses exquisite defense system and Allicin extraction several therapeutical properties, contributed by several sulfur- The outer skin of the garlic cloves was peeled and crushed in a garlic containing compounds, mainly allicin. Allicin (C3H5SS(O)C3H5) press. The pressed garlic was then collected in a beaker and mixed is an organosulfur compound, first isolated and studied in the thoroughly. 700-900 mg of the pressed mash was weighed and laboratory [1]. It is known as 2-propene-1-sulfinothioic acid S-2- transferred to a 50 ml centrifuge tube. Using a volumetric pipette, 25 ml propenyl ester, thio-2-propene-1-sulfinic acid S-allyl ester, diellyl of cold water was delivered to the sample and immediately capped and disulfide-oxide, diallyl thiosulfinate, and percent composition: shaken vigorously for 30 seconds. Heat transfer was avoided from C - 44.4%, H - 6.21%, O - 9.86%, and S - 39.52% [2]. The interaction hands by holding the tube cap while shaking. An additional 25 ml of of non-protein amino acid alliin with the enzyme alliinase produces cold water was added and shaken for 30 more seconds to dilute and mix allicin [3]. Alliin (S-allylcysteine sulfoxide, percent composition: the solution. Each sample is filtered through 0.45 µm glass filter into C - 40.66%, H - 6.26%, N - 7.90%, O - 27.08% and S - 18.09%) HPLC vial and capped for injection. consists of a sulfoxide group, allyl group and the amino acid cysteine (contains SH rather than S=O). Alliin is synthesized from S-Allyl HPLC conditions cysteine (deoxyalliin) [4]. It possesses a wide variety of medicinal Column: Phenomex ProdigyTM ODS (3), 5 µm, 100 Å, 4.6 mm × 250 mm, properties including antimicrobial, bacteriostatic, anti-thrombotic, mobile phase:methanol:water (50:50), flow rate: 1.0 ml/minutes, anti-fungal, anti-inflammatory, anti-cancer, and anti-atherosclerotic detector: 240 nm, injection Vol.: 25 µl, column temperature: 28°C, run activities along with the capacity to lower serum lipid levels and time: 16 minutes. ocular pressure [5-8]. On comminuting fresh garlic cloves, alliin is converted into diallyl thiosulfinate (allicin) in seconds. Allicin and Procedure the other thiosulfinates are unstable, but its stability can be greatly Garlic Pearls, a soft gel of Ranbaxy, was taken as the standard. An improvedby dissolving and diluting in water [9]. extraction solvent blank of water/methanol was prepared. A single injection was made of the blank. A single injection of the reference High-performance liquid chromatography (HPLC) has superior standard was also made. A linearity curve for allicin was prepared, resolving power due to the high-pressure separation condition, with the origin ignored, using peak versus concentration (µg/ml). making it the most preferred chromatographic technique for the Linear regression analysis on the data was performed. The coefficient separation of biological compounds. For the current study, the of determination, r2 concentration of allicin in the isolated fraction is standardized prepared was made. The percent allicin in the samples against allicin by HPLC analysis. Sample analysis is carried out by extraction of reference was calculated must [10]. be ≥0.999. A single injection of the sample garlic cloves with water and quantified against the isolated allicin external standard. Many researches are being carried out to improve Leukocyte culture method the current scenario of cancer. However, until now less or no study Chromosome preparations were obtained from phytohemagglutinin has been done to check the genotoxic effect of allicin. This current (PHA)-stimulated peripheral blood lymphocytes following the modified work is an attempt to examine the effect of allicin on human blood method of Hungerford [11]. About 2 ml of venous blood sample was leukocyte. collected in a sterile heparinized syringe. 0.5 ml of the blood was Nishu et al. Asian J Pharm Clin Res, Vol 8, Issue 6, 2015, 153-156 Fig. 1: High-performance liquid chromatography analysis of garlic pearls showing the retention times Fig. 2: High-performance liquid chromatography analysis of garlic extract showing the retention times inoculated into the vials containing 5 ml of RPMI 1640 medium containing 37°C after which it was centrifuged for 6 minutes. The supernatant 1 ml of FB serum and 0.2 ml of PHA under aseptic condition. The culture was carefully removed, and the cells were fixed with 6 ml of filtered vials were then placed in an incubator at 37°C. The cultures were shaken carnoys fixative (3:1 methanol: acetic acid). The tubes were left at room periodically, and carbon dioxide was released once every day. temperature for 2 hrs. One change of fixative was given prior to slide preparation. Treatment with standard garlic pearls At the 71st hrs of incubation, the cultures were treated with different Slide preparation concentration of standard Garlic Pearls (10, 20, 40, 80 µg/ml) for The cell pellet was suspended in a small quantity of freshly prepared 1 hrs. After 1 hrs of incubation, the culture was thoroughly washed by fixative. A test slide was prepared by gently placing a drop of the cell centrifuging the content at 1000 rpm for 10 minutes, and supernatant suspension on a cleaned glass slide and dried immediately on a hot were discarded carefully and 6 ml of fresh RPMI 1640 medium was plate (40°C). The test slide was examined under the microscope for added to the pellet and mixed well with Pasteur pipette. cell density and metaphase spreads. Other slides were prepared after suitable modifications. Treatment with extracted allicin st At the 71 hrs of incubation, the cultures were treated with different Staining procedure concentration of extracted allicin (10, 20, 40, 80 µg/ml) for 1 hrs. After The slides were stained in 4% Giemsa solution for 4 minutes and 1 hrs incubation, the culture was thoroughly washed by centrifuging the washed in distilled water for 1 minutes and then air-dried. content at 1000 rpm for 10 minutes, and supernatant were discarded carefully and 6 ml of fresh RPMI 1640 medium was added to the pellet Microscopic analysis and mixed well with Pasteur pipette. Scoring of well-spread and stained cells was performed under oil immersion (×100) of the light microscope (Leica ATC 2000). The well Culture harvesting spread metaphases were photographed using Olympus microscope After washing process, the dividing cells were arrested at metaphase by with the camera. adding two drops of 0.001% colchicine solution to each culture vial. The cultures were incubated further for 20 minutes at 37°C. The contents of RESULT AND DISCUSSION the vials were then transferred to 15 ml centrifuge tubes and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, and the The chromosomal aberration analysis is one of the widely used cells were resuspended in a small amount of solution left behind by parameters for testing the protective effects of natural compounds gently tapping the tube. 6 ml of pre-warmed (37°C) hypotonic solution on the drug and chemical induced toxicity. The modulatory effect of (0.075M KCl) was added to the tubes, and the contents were mixed natural compounds on the chromosomal aberration induced by various gently using a Pasteur pipette. It was then incubated for 5 minutes at kinds of chemicals and drugs are well established [12-15]. 154 Nishu et al. Asian J Pharm Clin Res, Vol 8, Issue 6, 2015, 153-156 HPLC technique was applied for the present research work. In 2012 although the proportion and amount of various constituents may differ Rahman et al. [9] reported that except HPLC none other technique is significantly [17,18]. Allicin liquid forms thiosulfinate compounds. reliable for the study of allicin. A study showed that the retention time These components are difficult to measure or detect without HPLC. of allicin seemed to vary from 3 to 3.7 minutes/ml in several garlic Hence, in this study we used high HPLC for determination of allicin in varieties [16]. Optimum conditions were applied for extraction of garlic. This study seems to be the one of the best methods to measure allicin from Indian garlic, and garlic oil was used as a standard. HPLC genotoxic effects in human leukocytes. result showed that the retention time of standard garlic pearls is 3 minutes/ml and that of garlic extract was noted to be 2.2 minutes/ CONCLUSION ml (Fig.